Stem cells have multi-differentiation potency to differentiate into cells of various tissues by specific differentiation-inducing stimuli as well as self-renewal capacity at the in undifferentiation stage. They are divided into embryonic stem cells (ES cells) and adult stem cells depending on their differentiation potency and time to be generated. ES cells are isolated from the inner cell mass (ICM) of embryos at the blastocyst stage. ES cells include three types of germ layers, i.e., endoderm, mesoderm and ectoderm, and are pluripotent cells that are capable of differentiating into virtually every type of cells found in an organism. However, there still remain difficulties involved in how to control their differentiation potency as well as the problem of ethics.
In contrast, adult stem cells appear at the stage of organ formation during the embryonic development or at the adult stage. They are organ-specific and multipotent, i.e., they are generally committed to give rise to cells constituting a specific organ. These adult stem cells remain in most of adult organs and perform the critical role of continually replenishing the loss of cells occurring normally or pathologically.
Representative adult stem cells include hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) present in bone marrow. HSCs give rise to various blood cells such as erythrocytes, leukocytes and thrombocytes; and MSCs, to the cells of mesodermal tissues such as osteoblasts, chondroblasts, adipocytes and myoblasts.
Since the successful isolation of human embryonic stem cells or adult stem cells was reported, clinical applications of the stem cells have drawn increasing interests. The most noticeable potential application of the stem cells is their use as a cell supply source for cell replacement therapy. Hard-to cure diseases, e.g., neurodegenerative disease such as Parkinson's and Alzheimer's diseases, quadriplegia resulting from spinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiac infarction, hepatocirrhosis and other chronic diseases, are caused by the disruption and permanent functional disorder of the cells constituting certain organs. Cell replacement therapy by which the loss of cells is replenished from the outside has been presented as a promising remedy therefor.
ES stem cells can be obtained from bone marrow, and it has been reported that HSCs, MSCs and multipotent adult progenitor cells (MAPCs) exists in bone marrow. Several reports have demonstrated that MAPCs derived from bone marrow can differentiate into cells of other tissues such as nerve cells, endothelial cells and hepatocytes as well as into osteoblasts, chondroblasts and adipocytes similar to MSCs (Reyes M, et al., Blood 98: 2615-2625, 2001; Reyes M, et al., J. Clin. Invest. 109: 337-346, 2002). However, notwithstanding the remarkable effect expected of the cell replacement therapy using bone marrow-derived stem cells, there exist many limitations in its clinical applications. For example, the conventional method for isolating stem cells from bond marrow has the problem of requiring several steps of complicated operations, which may impose mental and physical stress on a donor. Further, it is very difficult to find a donor for bone marrow transplantation who has an antigen phenotype identical to a recipient.
Since the presence of MSCs in bone marrow was discovered by Friedenstein (Friedenstein A J, Int. Rev. Cytol. 47: 327-345, 1976), there have been numerous studies on their differentiation potency and use as a cell therapeutic agent. Especially, the clinical use of MSCs for the treatment of cartilaginous diseases is in the process of regulatory approval, and a therapeutic agent comprising the same for treating osteocyte-relating diseases is about to enter a clinical stage. However, MSCs in bone marrow have a limitation in their applicable targets due to their restricted differentiation and proliferation potencies, and they are still not free from the previously reported problems in obtaining bone marrow-derived stem cells. Further, MAPCs derived from bone marrow show a wide applicable range in terms of differentiation potency, but they also have problems in how to reproducibly isolate and cultivate besides the limitations imposed by the bone marrow origin.
Meanwhile, as it has been reported that umbilical cord blood contains a large quantity of stem cells and is a source of HSCs, there have been made several attempts to clinically remedy blood disorders by cord blood transplantation. Further, cord blood transplantation triggers a much lower degree of graft-host rejective interaction than bone marrow transplantation, and extensive studies for its clinical use have been carried out.
However, there still remain problems in the isolation and cultivation of MSCs in cord blood (Erices A, et al., Br. J. Haematol. 109: 235-242, 2000; Lee O K, et al., Blood 103: 1669-1675, 2004; Wexler S A, et al., Br. J. Haematol. 121: 368-74, 2003). Also, there is no report on a reliable method for isolating and culturing stem cells capable of differentiating into various types of cells such as neurons, osteoblasts, myoblasts, adipocytes and so on from cord blood.
The present inventors have endeavored to develop a method for obtaining multipotent progenitor/stem cells that can be effectively used for cell therapy, cell replacement therapy, an organ restoration technique, or an organ production, and have established effective methods for isolating and culturing multipotent progenitor/stem cells from mononuclear cells derived from cord blood, and differentiating the multipotent progenitor/stem cells into various types of cells such as neurons, osteoblasts, myoblasts, endothelial cells, hepatocytes and dendritic cells.